The Molecular Imaging Branch (MIB) aims to exploit positron emission tomography (PET) as a radiotracer imaging technique for investigating neuropsychiatric disorders, such as autism, depression, addiction, schizophrenia, and Alzheimer's Disease (AD). Fundamental to this mission is the development of novel radioactive probes (radiotracers) that can be used with PET to measure changes in low level proteins in the brains of living human subjects where these proteins are suspected to have critical involvement in the progression of neuropsychiatric disorders. Such proteins include some neuroreceptors, transporters, enzymes, and plaques. PET is uniquely powerful and sensitive when coupled with the use of biochemically-specific PET radiotracers. The chemical development of new radiotracers is the key to exploiting the full potential of PET in neuropsychiatric research. However, a successful PET radiotracer must satisfy a wide range of difficult-to-satisfy chemical, biochemical, and pharmacological criteria. Consequently, PET radiotracer development is highly challenging. In fact, our research has some parallels with drug discovery in that it entails high effort and heavy risk but can reap rich biomedical rewards. As of now, the number of potentially interesting PET imaging targets (brain proteins) far exceeds the range of currently available and useful radiotracers. Within MIB, the PET Radiopharmaceutical Sciences Section (PRSS) places a concerted effort on all chemical and radiochemical aspects of PET radiotracer discovery. Our laboratories are equipped for medicinal chemistry and automated radiochemistry with positron-emitting carbon-11 (t1/2 = 20 min) and fluorine-18 (t1/2 = 110 min). These two very short-lived radioisotopes are available to us daily from the adjacent cyclotrons of the NIH Clinical Center (Chief: Dr. P. Herscovitch). Our Section interacts seamlessly with the Section on PET Neuroimaging Sciences in our Branch (Chief: Dr. R.B. Innis) for early evaluation of potential radiotracers in biological models and in animals. Subsequent PET research in human subjects is also performed in collaboration with the Imaging Section under Food and Drug Administration oversight through 'exploratory' or 'full' Investigational New Drug applications. In the period covered by this report, we worked on developing and producing PET radiotracers for several protein targets. These include translocator protein 18 kDa (TSPO); the NR2B sub-site of the NMDA receptor, serotonin subtype 1B (5-HT1B)receptors, dopamine subtype-3 (D3)receptors; and several enzymes (COX-1, COX-2, PDE1 and PDE4 subtypes, and OGA). One radiotracer that we earlier developed for TSPO imaging (C-11PBR28) has been applied by many imaging centers to investigate brain inflammatory conditions (i.e., neuroinflammation) in response to various neurological insults (e.g., stroke, epilepsy and neurodegeneration). An unexpected finding is that healthy human subjects, because of a genetic difference, carry either one or both of two distinct forms of TSPO and that these interact differently with C-11PBR28, complicating the analysis of PET studies. Consequently, we sought to develop genetically-insensitive TSPO radiotracers. We explored new chemotypes with potential to provide superior PET radiotracers for TSPO. One of our new radiotracers, C-11ER176, appeared promising for avoiding genotype sensitivity based on our evaluation in animals and in human tissue in vitro. In addition, while C-11ER176 does not show the expected genotype insensitivity in living humans, it does turn out to be one of the highest performing TSPO radiotracers yet known, and especially is able to quantify TSPO in all subjects of identified genotype. We and other imaging centers now plan to use this radiotracer for clinical studies in human subjects. C-11ER76 production has been established in a new cGMP facility for this purpose. We are also developing longer-lived and therefore more broadly useful F-18 labeled versions of this radiotracer. We are now developing radiotracers for other targets relevant to the study of neuroinflammation, such as the cyclooxygenase (COX) subtype 1 and subtype 2 enzymes. Very promising C-11 labeled radiotracers have been identified, and two of these are now being advanced for evaluation in human subjects. These radiotracers may provide more biochemical and cellular specificity for investigation of neuroinflammation. They can prove useful for the development of improved anti-inflammatory drugs. Receptors acted upon by the important neurotransmitter glutamate are of interest for the study of addiction and other disorders, notably Fragile X syndrome associated with autism spectrum disorder, and schizophrenia. Metabotropic glutamate (mGlu) receptors are examples. We have reported our comparison of two new high-performing mGlu5 radiotracers (C-11SP203 and C-11FPEB) in human subjects and our recently developed radiotracer (F-18FIMX) for a related receptor target, mGlu1. Studies in human show this radiotracer performs exceptionally well. NMDA, another protein acted upon by glutamate, is strongly implicated in the pathogenesis of schizophrenia. Our research has led to promising radiotracers for imaging the NR2B binding site on the NMDA receptor. PET radiotracers can provide important quantitative information on experimental therapeutics for neuropsychiatric disorders, such as ability to cross the blood-brain-barrier and to engage with a target protein. In collaborations with academia and Pharma, we are developing several radiotracers for this purpose (e.g. radiotracers for the NR2B site). Some of these radiotracers are targeted at proteins not previously been imaged in living human brain that may have eventual clinical research utility. These proteins include subtypes of phosphodiesterase (especially with regard to depression), and the enzyme OGA (especially with regard to dementia). We are currently evaluating a very promising radiotracer for OGA in human subjects. The development of radiotracers for phosphodiesterase subtypes shows early promise and one radiotracer candidate is being progressed to human evaluation. We are advancing our methodology for improved radiotracer development. New labeling agents have been developed for C-11 chemistry, notably C-11fluoroform, which opens up wider chemical space for potential radiotracer development. We have also succeeded in developing a method for F-18fluoroform synthesis at a higher molar activity than hitherto possible, again expanding possibilities for new radiotracer development. New radiolabeling chemistries continue to be developed to utilize C-11fluoroform or F-18fluoroform for preparing new radiotracers. Sensitive mass spectrometry has been developed to measure radiotracer concentration in blood following intravenous administration, as is required to analyze PET experiments. LC-MS/MS has the potential to avoid the demanding logistics associated with measuring fast-decaying radioactivity, and we now plan to implement this methodology. Productive collaborations continue with external academic chemistry and medicinal chemistry laboratories, nationally and internationally, and with pharmaceutical companies. Productive collaborations also exist with other centers working with PET and its associated radiochemistry and radiotracer development. The laboratory is active in training new scientists for this field at graduate and postdoctoral level. We produce some useful radiotracers that have been developed elsewhere for PET investigations in animal or human subjects e.g., C-11rolipram (for PDE4 enzyme imaging), and C-11clozapine for investigation of DREADD technology. Each PET experiment requires a radiosynthesis of the radiotracer on the same day, and hence radiotracer production is a regular activity.